Resonance and Pre-resonance Raman Spectra of the Antitumor Drugs Adriamycin and Dalnnomycin

Michel Manfaitt and Lucien Bernard Laboratoire de Recherches Optiques, UER Sciences Exactes et Naturelles, BP 347, 51062 Reims Ctdex, France

Theophile TheophanidesP UniversitC de MontrCal, Dtpartement de Chimie, CP 6210 Montrkal, Qukbec, Canada H3C 3V1

Resonance and pre-resonance Raman spectra of solids, aqueous and D20 solutions of adriamycin and daunomycin are reported. The Raman bands are due to the chromophore anthraquinone and are coupled with the first electronic m-m* transition of the molecules. All the observed Raman bands have been assigned to vibrations of the chromophore. Resonance enhancement for most of the Raman bands has been observed in relation to the m-n* transition. Spectra taken at different pH values show interesting Raman intensity changes owing to a shift of the m-m* transition towards longer wavelengths. An n-m* transition has been assigned to the shoulder near 500 nm of the absorption spectra of the molecules on the basis of the intensity changes of some Raman bands involving the carbonyl vibrations.

INTRODUCTION

Adriamycin' and daunomycin* (Fig. 1) are both gly- cosidic anthracycline antibiotics and are known to be anticancer agents used in the treatment of acute leuke- mia and solid tumors in man.316 The biological activity of these drugs seems to be due to a complexation with DNA and the interaction is thought to be through intercalation of the chromophore with the base pairs of DNA.7-9 Although considerable interest has been centred towards the therapeutic effectiveness, these drugs, however, do possess several toxic effect^,^ such as stomatitis, cardiotoxicity and digestive disturbances. The investigation of the molecular details of the binding of these drugs to DNA or to metal ions in connection with their skeletal toxicity will be reported in a later publication in which the anthracycline chromophore will be used as a probe to study the drug-DNA inter- a c t i o n ~ . ~ ' ~ " The chromophore is assumed to be the 7~

system of the quinonoid structure which absorbs

0 OH

OH

Figure 1. Structure of adriamycin (R =OH) and daunomycin (R = H). t Authors to whom correspondence or reprint requests should be addressed.

strongly in the visible. The visible spectra of adriamycin and daunomycin are identical with absorptions near 480 and 500 nm. Although the pre-resonance Raman spec- tra of adriamycin in relation to the first electronic band at 480nm has been published," yet complete band assignments were not given. The present investigation includes a study of the vibrational assignments of the resonance Raman bands of the chromophore in both adriamycin and daunomycin molecules and the overlap of several bands in solution at varying pH values together with excitation profiles and intensity changes of the Raman bands as a function of excitation wavelength. The studies were confined to the effect of the chromo- phore with its carbonyl and OH groups on the positions of the bands. Preliminary resonance Raman results have been previously reported on adriamycin12 and on both adriamycin and daunomycin by us. 13,14

EXPERIMENTAL ~

Adriamycin and daunomycin hydrochlorides (Farmi- talia, Milan, and Roger Bellon Laboratory, Paris) were used as received. Solid spectra were obtained as powders with the spinning technique. Solution spectra in distilled water or deuterium oxide were obtained in quartz 10 x 10 mm cells at various pHs. The low concentration of the order of 1 0 p 5 ~ is ideal for in vivo experiments in biological systems. The aqueous solutions were not buffered and only fresh solutions were recorded in order to avoid any decomposition from the laser beam. During all the experiments it was verified that laser irradiation had no effect on the visible absorbance spectra of the samples in aqueous solution. NaN03 was added in the solutions and powders as an internal standard. The Raman line corresponding to the vibrational mode u,NOi (1070 cm-' in the solid state and 1052 cmpl in aqueous solution) is taken as reference for the measures in intensities.

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RAMAN SPECTRA OF THE ANTITUMOR DRUGS ADRIAMYCIN AND DAUNOMYCIN

Resonance and pre-resonance Raman spectra were recorded with a Model T 800 Coderg spectrometer with a triple monochromator and a cooled EM1 9558 QB photomultiplier. A Spectra Physics 164 Art ion laser with excitation lines 457.9,472.6,476.5,488,496.5 and 501.7nm was used. The laser was equipped with a tunable dye, Coherent Radiation Model 490 which provided the line 615 nm for the pre-resonance spectra. The power of the lines was varied from 20 to 200 mW. A small two prism monochromator (Anaspec 300 S) was used to remo\= the background plasma lines. The Raman frequencies are reported with *2 cm -' accuracy and the intensities are measured by the peak heights of the bands, which are corrected for instrumental response and Raman scattering.

The depolarization data were obtained with a polarizer and a scrambler. The spectra were taken with a slit width of 4 and 6 cm for the solid and the solutions, respectively. The absorption spectra were obtained with a Beckman 5240 spectrometer, whereas the fluores- cence spectra were obtained with the T 800 Coderg spectrometer.

RESULTS AND DISCUSSION

Absorption and fluorescence spectra (laser excitation wavelength 457.9 and 488nm) are shown in Fig. 2 together with the positions of the excitation lines on the

476.51 488 472.6

,i,L,,, ,501.7

t

nm

Figure 2. Absorption spectra of adriamycin (a) and daunornycin (a') in H20 at pH = 6.5. Fluorescence spectra of adriamycin in H20 (b -; d 01, solid (c . . .; e 7 ) and daunornycin in H20 (b' -; d' 0) and solid (c' . . .; e' V). Laser excitation wavelength AD =457.9 nm for b, b', c, c' and 488 nrn for d, d', e, e'.

t i -u I

I600 1200 860- 400 I

cm- '

Figure 3. Resonance Raman spectra f f (a) adriamycin (2.6~ I V 4 ~ ) and (c) daunomycin (4x10- M), AD=457.9 nm. h e - resonance Raman spectra of (b) adriamycin (5.2 X M) and (d) daunomycin ( ~ X I O - ~ ) , Xo=615 nrn.

absorption band. Comparative pre-resonance and resonance Raman spectra of adriamycin and daunomy- cin are shown in Fig. 3 . Remarkable intensity changes are taking place as we go from conditions of pre- resonance to resonance. The resonance Raman spectra in solid state, water and deuterium oxide are shown in Figs 4 and 5 together with depolarization spectra in water. All Raman vibrations are planar and are polarized (see Table 1). Intensity enhancement and excitation profiles of Raman bands have been obtained in the regions 615 to 457.9 nm and 457.9 to 501.7 nm and are shown in Figs 6 and 7. The pH dependence of the Raman bands and electronic absorption bands is shown in Fig. 8. The assignments of the Raman bands of the chromophore are given in Table 1.

The sample concentration of the solutions was of the order of 10 -4 to 1 OP5 M which is quite low compar:d to concentrations of ordinary Raman spectra (10- M). These experimental conditions are suitable for in uivo studies of antibiotics with chromophoric groups. However, under these conditions one sees only the chromophore vibrations which is part of the total mole- cule. No bands can be attributed to the lateral groups, i.e. the glycosidic moiety or the saturated ring with their functionalities. The vibrations of these parts can be obtained in Raman only if we excite the molecules in the UV or in the red (with high laser power) to avoid resonance conditions with the chromophore. However,

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M. MANFAIT. L. BERNARD AND T. THEOPHANIDES

. I t

i 600 I200 800 400

c m Figure 4. Resonance Raman spectra of adriamycin, h o = 457.9 nm: (a) solid; (b) H20 solution Ill ( 2 . 6 ~ M); (c) H20 solution I, (2.6x M); (d) D20 solution /II ( 2 . 6 ~ M).

when taking the spectra one must also avoid the fluorescence spectrum which is very intense (see Fig. 2).

Wavelength dependence and intensity variations of some Raman bands of adriamycin and daunomycin

A quick glance at the spectra shows that the Raman bands observed here are only due to the quinonoid chromophore of the two drugs and that they are coupled to the T-T* electronic transition near 21 000 cm-' (477 r~m)"-*~ giving rise to pre-resonance (615 nm) and resonance (501.7-457.9 nm) Raman spectra. The vibrations of the aromatic ring modes (rings A, B and C ) of type g are coupled with the T-T* electronic transition of the quinonoid structure.

The intensity changes of the resonance Raman bands of adriamycin and daunomycin as a function of wavelength variation (615-457.9 nm) are shown in Fig. 6. From the drawings it is shown that as one approaches the absorption maximum from 615 nm going towards 457.9 nm some bands are more enhanced than others. The bands that are most enhanced in intensity are the 1587 andP94 cm-' for adriamycin and 1587, 994 and 1310 cm- for daunomycin. These bands are associated with the IT system of the quinonoid s t r u ~ t u r e ' ~ ' ' ~ (see Table 1, assignments) and coupled to the TIT* elec- tronic transition of the molecules. The least coupled bands are those which show small enhancement of intensity (352, 444, 465 and 1084 cm-* for adriamycin

4 I600 1200 800 400

cm-l

Figure 5. Resonance Raman spectra of daunomycin, h o = 457.9 nm: (a) solid; (b) H20 solution 111 ( 4 x M); (c) H20 solu- tion I, ( 4 x M); (d) D20 solution /I, (4X MI.

. -

and 352,447,465 and 1086 cm-' for daunomycin). It is reasonable to assume that these bands are the least coupled to the n-z* transitions of the molecules and are assigned to the carbonyl and ring bendings, whereas the 1084cm-' is assigned to the C-0 of the methoxy group (see Table 1, assignments). The carbonyl band at 1642 cm-' of adriamycin is moderately enhanced in intensity indicating some degree of coupling with the C=C and the T system of the quinonoid structure.

Partial excitation profiles of some Raman bands for both drugs in the wavelength region 457.9-501.7 nm are shown in Fig. 7. As one approaches the absorption band at 501.7 from 457.9 nm the Raman bands in the range of 444-467 cm-' that are most affected are those involving the carbonyl bendings which are coupled with the ring bending motions. The Raman bands assigned to the ring breathing, C=C and C-C stretches are less affected in intensity. This behavior is expected if the absorption at 480nm is assigned to a FIT* transition of the quinonoid structure16-" and the band near 500 nm to a n - ~ * transition. The intensity profiles then of the Raman bands can be explained on the basis of this IT-IT*

and n - ~ * assignments and a vibration-electronic coup- ling. This coupling is also indicated from pH experi- ments (see later). Unfortunately, we were unable to obtain energy profiles for the Raman bands of adriamy- cin in water solutions due to its strong fluorescence band in water shifted to the blue with respect to the solid spectrum and to that of daunomycin (Fig. 2). This shift allows the excitation of the fluorescence spectrum which of course is much stronger than the Raman spectrum and does not permit Raman measurements.

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RAMAN SPECTRA OF THE ANTITUMOR DRUGS ADRIAMYCIN AND DAUNOMYCIN

30

20 K

10

20

K

10

b )

cm-'

Figure 6. Intensity increases of some Raman bands of (a) adria- mycin and (b) daunomycin in H20 as a function of wavelength excitation, Xo = 61 5-457.9 nm.

Effect of pH on the absorption spectra and the Raman band inlensities

The effect of pH on the electronic absorption spectra and the Raman bands of adriamycin is shown in Fig. 8. Increasing the pH of the water solutions from 6.7 to 10.14 caused considerable changes in the ab~orp t ion '~ and resonance Raman spectra. The changes were more drastic with daunomycin which does not have the lateral secondary OH groups. The intensities of the Raman

( b )

44Yi

457.9 472.6 488 501.7

Excitation (nm)

Figure 7. Excitation profiles of some Raman bands of solid (a) adriamycin and (b) daunomycin (the intensities were measured and normalized with respect to the NO, symmetric stretch (1070 cm-')).

nm

9.18 9.69

994

1215

- -.-

. . . . , . . . . . . . 444 cm-'

- 994

1215 -.-

1.9

,I4

I I 1 -

6 7 8 9 10

PH

Figure 8. Effect of pH on the absorption spectrum (a) and on some Raman bands (b) of adriamycin.

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M. MANFAIT. L. BERNARD AND T. THEOPHANIDES

Table 1. Resonance Raman bands of adriamycin and daunomycin recorded with 457.9 nm excitation line in solid state, water solution and D208

Adriamycin Daunomycin

Solid

100 vw 186 vw 320 vw sh 347 w 433 sh 447 m 467 m

591 vw

920 v w

992 vw

1086 vw 1120vw 1153vw 1209s 1239 s 1300 m 1327 m 1348 sh 1386 sh

1410sh 1438 s 1458sh 1581 m 1646 vw 1676 vw

HzO PH = 6.48 D,O pD = 6.5

352 vw 437 s h 444 m 465 m

595 vw 824 vw

921 vw 963 vw 994 vw

1070 vw 1084 vw 1115vw 1152vw 1215s 1245 s 1308 m 1345 w

1420 sh 1445 s 1457 sh 1587 m 1642 vw

319vw 350 v w 433 sh 446 m 461 m

590 vw 822 vw

897 v w

964 m

1060 vw 1083 vw

1227 v w 1250 vw 1284 vw 1337 s

1408 sh 1423 s

1462 s 1552 w

Solid

350 vw 433 sh 449 m 466 w

920 vw

990 vw

1084 vw

1153vw 1210s 1249 s 1300 m 1325 s 1347 sh

1413 m 1440 s 1458 sh 1577 m 1642 vw 1673 vw

H,O pH = 6.48 D,O pD= 6.5

352 vw 435 sh 447 m 465 w 479 sh 620 vw 822 vw 852 vw

922 vw 964 vw 994 vw

1072 vw 1086 vw

1157vw 1214s 1246 s 1310 m 1345 w

1420 sh 1448 s 1457 sh 1587 m 1647 vw

330 sh 350 vw 437 sh 447 m 462 w

624 vw 824 vw

902 vw

967 m

1089 vw

1224 v w 1252 vw 1283w 1334sh 1344 s I 1418s

1463 s 1553 w

Assignment

6(C=O) bending coupled to ring bending

ring bending coupled to the (C=O) ring bending coupled to the (C-0)

S(C-0-D) (1152/897=1.28)

ring breathing/s(C-O-D) (1 219/964 = 1.26)

u(C-0, methoxy)

6(C-0-H) v ( C ~ 0 ) + 6(C-0-H) + 6(C--H) u(C-O)+S(C-O-H)+S(C-H)

v(C-C)+v(C-O)

v(C=C)+ v(C-C)+ v(C=O...H(D))

u(C=C) + v(C=O...H(D)) u(C=O...H(D)) hydrogen-bonded v(C=O) free

a v, stretch; A, bending; D, deuterium; depolarization ratio p was in the range 0.3-0.5 for all the bands.

Chromophore :

bands of adriamycin decreased rapidly around pH = 9 and at higher pHs the resonance Raman spectrum is completely lost. The intensity decrease of the Raman bands may be related to the red shift of the band assigned to a FIT* transition of the quinonoid struc- ture." The red shift is expected on successive proton dissociation (deprotonation) of the intramolecularly hydrogen bonded phenolic hydroxyl groups, and the quinonoid system may be more delocalized including the carbonyl and phenolic oxygens. The intensities of the Raman bands may be affected because of the red shift and the structural changes taking place as a result of the two proton dissociations of the phenolic hydrogens as

follows: H2(chromophore)t-+HC + H(chromophore)- H(chromophore)--H' + (chromophore)

The Raman intensities of the bands which were cou- pled to the IT-IT* transition at 480 nm decreased more rapidly than those which were assumed to be coupled to the IT* transition at 500 nm (see Fig. 8). It is reason- able to assume that destruction of the intramolecular hydrogen bond giving rise to the above two ionization states is responsible for the Raman intensity changes. It is interesting to notice that frequency shifts do occur in the S(C=O) and S(C-0) bands at 444 and 465 cm-l.

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These two bands at higher pH values (9.9) decrease i: intensity and cdalesce into one band at about 470 cm- corresponding most likely to one type of carbonyl" (C=O):

From Fig. 8 we see that the intensities of the Raman bands at 1587, 994 and 1308 cm-' related to the delo- calized system are most affected with pH increases, whereas the carbonyl bands are less affected in intensity, but also slightly affected in frequency as well.

Assignment of the Raman bands

The assignment of the Raman frequencies of the chromophore is given in Table 1. The resonance Raman spectra in water, D 2 0 solutions and solids allow assignment of the phenolic-OH vibrations, which are completely deuterated in D20. The assignments made by Hillig and Morris" on the basis of pre-resonance Raman spectra obtained with the 632.8 nm He-Ne excitation line were helpful in completing our results of the resonance spectra. Pre-resonance spectra were obtained here only with one line 615 nm which lies near the fluorescence band (see Fig. 2) and is the only one we could use with our tunable dye. The pre-resonance spectra are helpful for the assignment of some Raman bands of the chromophore

The bands due to the u(C=O) vibration are readily assigned to the weak bands at 1676cm-' (free) and 1646 cm-' (hydrogen-bonded) for adriamycin an: to the bands at 1673cm-' (free) and 1642cm- (hydrogen-bonded) in daunomycin in the solid state (see Table 1). As expected, in this type of the hydroxyl group is hydrogen-bonded to the C=O group. The position of the Raman band of the hydrogen chelated C=O is shifted to lower frequencies, parti- cularly in the D 2 0 solutions, where it seems to be coupled with the ring C=C stretching vibrations in the region 1580-1450 cm-'. In D2O solutions, where the phenolic hydrogen is replaced by deuterium this coup- ling seems to be stronger. The broad Raman bands in the region 1445 cm-' for adriamycin and 1448 cm-' for daunomycin on deuteration split into two, 1423 and 1462 cm-' and 1418, 1463 cm-' and are assigned to C=C and C-C stretches of the aromatic carbons (see Table 1). These bands shift only slightly on deuteration of the phenolic hydroxyl groups indicating some coup- ling also with C=O=H(D) frequency. The broad band in the region of 1300 cm-' on deuteration seems to shift to 1284 and 1337 cm-' and 1283 and 1344 cm-' for adriamycin and daunomycin, respectively. These bands are more affected on deuteration than the previous ones

and are assigned to u(C=O) and u(C=C) vibrations. The u(C-0) vibration is directly affected on deutera- tion. The bands in the region 1115-1240 cm-' are affected the most on deuteration and must involve the hydroxyl bendings. These bands are assigned (see Table 1) to a mixture of 6(C-Ol-H) and u(C*==O) vibrations. The new band at 897 cm- in D20 solutions may be due to an isotopic shift of these vibrations on deuteration (uH/uD= 1.28-1.34). However, in this region one must also expect the 6(C-H) vibrations which may be the weak bands at about 1225cm-' observed after deu- teration. The weak bands at about 1060 and 1080 ern--' may be attributed to the methoxy carbon-oxygen stretching frequency. The band near 990 cm-' shifts to a broader band at 964 cm-' in D2O and is attributed to the ring breathing motion of the quinonoid structure. This band is perturbed on deuteration of the OHs and may be a mixture with 6(C-0-D) (see Table 1). The low frequency bands in the region 350-460 cm-' are the least perturbed in D 2 0 solutions. The 350 cm-' band is attributed",16 to the carbonyl bending vibration 6(C=O), weakly coupled to the ring bending motion, whereas the pair of bands near 440 and 460cm-' is assigned to a ring bending coupled to 6(C=O) and 6(C-0) bending carbonyl motions, respectively.

CONCLUSION

In summary, it is found in this study that resonance Raman spectra can be used to follow the chromophore and its most sensitive vibrational bands in a biologic milieu on varying the pH of the solutions and the resonance conditions. The resonance Raman spectra, excitation profiles and intensity enhancement have been useful also for the assignment of the Raman bands of the chromophore. This Raman study was an attempt to learn more about the anthraquinone chromophore in these two antibiotic antitumor drugs in order to use it as a probe in our future in vitro and in vivo studies of the interactions of adriamycin with DNA.'."' Experiments are underway at the cellular level with the L1210 malignant cells. Furthermore, these drug-cell inter- actions take place at low doses23 of the drug of the order

M which is another advantage of using resonance Raman spectroscopy with these chromophoric drugs.

Acknowledgements

Adriamycin and daunomycin were a gift from Professor F. Arcamone's Laboratory (Farmitalia, Milan). We wish to thank Pro- fessor Arcamone and Dr Cassinelli for the generous samples of the drugs. We also wish to thank Dr Desbordes (Roger Bellon's Labora- tory) who generously supplied us with a sample of adriamycin. In addition, we are thankful to Drs Rimbault and Pierrard (University of Reims) for recording the absorption spectra and to the 'CoopCration France-Quebec'.

REFERENCES

1. F. Arcamone, G. Cassinelli, G. Franceschi, S. Penco, C. Pol, S. Radaelli and A. Selva, in International Symposium on Adriamycin, ed. by S. K. Carter, A. D. Marzo, M. Chione, I . H. Krakoff and G. Mathe. Springer-Verlag, Berlin, Heidel- berg, New York (1972).

2. R. Anguili, F. Arcamone, E. Foresti, N . W. Isaacs, 0. Kennard, W. D. S. Motherwell, L. Riva di Sanserverino and D. L. Wamper, Nature London New Biol. 234'78 (1971).

3. C. Tan, H. Tasaka, K. P. Yu, M. L. Murphey and D. A. Karnof- sky, Cancer 20,333 (1967).

@ Hcyden & Son Ltd, 1981 JOURNAL OF RAMAN SPECTROSCOPY, VOL. 11, NO. 2, 1981 73

M. MANFAIT, L. BERNARD AND T. THEOPHANIDES

4. 0. M. Young, Cancer Chemother. Rep. 3, 159 (1975). 5. F. Arcamone, Lloydia 40,45 (1977). 6. J. R . Brown, frog. Med. Chem. 15, 125 (1978). 7. W. J. Pigram, W. Fuller and L. D. Hamilton, Nature London

NewBiol. 235, 17 (1972). 8. S. Neidle and G. Taylor, Biochim. Biophys. Acta 479, 450

(1977). 9. M. Manfait, A. J. P. Alix, P. Jeannesson, J. C. Jardillier and

T. Theophanides, Seventh International Raman Conference Ottawa, 4-10 August, 1980, Canada.

10. M. Manfait, A. J. P. Alix, P. Jeannesson, J. C. Jardillier and T. Theophanides. unpublished.

11. K. W. Hillig II and M. D. Morris, Biochem. Biophys. Res. Commun. 71,1228 (1976).

12. B. P. Gaber, lnt. Lab. May/June, 1 1 (1977). 13. T. Theophanides, in Proceedings of the Sixth International

Raman Conference, ed. by E. D. Schmid et al., Vol. 1, p. 115 (1978).

14. M. Manfait, M. Berjot, L. Bernard and T. Theophanides, Proceedings of the Sixth lnternational Raman Conference, ed. by E. D. Schrnid eta/., Vol. 2, p. 150 (1978).

15. F. Arcarnone, G. Cassinelli, G. Fantini, A. Grein, P. Orezzi, C. Pol and C. Spalla, Biotechnol. Bioeng. 11, 1101 (1969).

16. E. A. Gastilovitch, G. T. Kryuchkova and 0. N. Shigorin, Opt. Spekfrosk. 38, 282 (1975).

17. I. M. Issa, R. M. Issa, M. S. El-Ezabey and Y. 2. Ahmed, Z. Physik. Chem. 242,169 (1969).

18. 2. Yoshida and F. Takabayashi, Tetrahedron 24, 913 (1968). 19. A. Kuboyama and K. Wada, Bull. Chem. SOC. Jpn 39, 1874

20. V. h p h n and J. Vodehnal, Coll. Czech. Chem. Commun. 36,

21. J. Rasanen and F. Stenman, Phys. Fenn. 10, 183 (1975). 22. R. M. Issa, Y. 2. Ahrned and A. H. Zewail, Egypt J. Chem. 15,l

23. S. R. Byrn and G. R. Dolch, J. Pharm. Sci. 67,688 (1978).

(1966).

3964 (1971).

(1972).

Received 7 July 1980

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